CHARACTERIZATION OF GLYCOSAMINOGLYCAN-BINDING DOMAINS PRESENT IN INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-3

Matrix metalloproteinase 3 cleaves insulin-like growth factor-binding protein 3 (IGFBP-3) into six fragments, four of which bind heparin-Sep harose (Fowlkes, J. L., Enghild, J. J., Suzuki, K., and Nagase, H. (19 94) J. Biol. Chem. 269, 25742-25746). Sequence analysis of IGFBP-3 hep arin-binding fragments shows that all fragments contain at least one o f two highly basic, putative heparin-binding consensus sequences prese nt in IGFBP-3. Epitope-specific antibodies generated against synthetic peptides containing these domains recognized IGFBP-3, yet were signif icantly inhibited from binding in the presence of heparin, demonstrati ng that these regions of IGFBP-3 contain functional heparin binding do mains, IGFBP-3 peptides containing one of the two heparin-binding cons ensus sequences bound heparin in a solid phase binding assay in a dose -dependent and saturable manner. However, the IGFBP-3 peptide containi ng the heparin-binding consensus sequence (KK)-K-149-GHA(153) bound he parin with similar to 4-fold less affinity than the IGFBP-3 peptide co ntaining the longer heparin-binding consensus sequence (219)YKKKQCRP(2 26). Examination of several well characterized glycosaminoglycans to i nhibit the binding of heparin to both heparin-binding IGFBP-3 peptides revealed that the most potent inhibitors were heparin, heparan sulfat e, and dermatan sulfate; chondroitin sulfate A and hyaluronic acid wer e intermediate in their inhibitory activities; and chondroitin sulfate C caused no inhibition, These studies identify and characterize the g lycosaminoglycan-binding domains in IGFBP-3, providing a basis for the better understanding of IGFBP-3 glycosaminoglycan interactions at the cellular and extracellular interface.